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The Effects of Melatonin on Colonization of Neonate Spermatogonial Mouse Stem Cells in a Three-Dimensional Soft Agar Culture

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The Effects of Melatonin on Colonization of Neonate Spermatogonial Mouse Stem Cells in a Three-Dimensional Soft Agar Culture Navid et al. Stem Cell Research & Therapy (2017) 8:233 DOI 10.1186/s13287-017-0687-y RESEARCH Open Access The effects of melatonin on colonization of neonate spermatogonial mouse stem cells in a three-dimensional soft agar culture system Shadan Navid1, Mehdi Abbasi1* and Yumi Hoshino2 Abstract Background: Melatonin is a pleiotropic hormone with powerful antioxidant activity both in vivo and in vitro. The present study aimed to investigate the effects of melatonin on the proliferation efficiency of neonatal mouse spermatogonial stem cells (SSCs) using a three-dimensional soft agar culture system (SACS) which has the capacity to induce development of SSCs similar to in vivo conditions. Methods: SSCs were isolated from testes of neonate mice and their purities were assessed by flow cytometry using PLZF antibody. Isolated testicular cells were cultured in the upper layer of the SACS in αMEM medium in the absence or presence of melatonin extract for 4 weeks. Results: The identity of colonies was confirmed by alkaline phosphatase staining and immunocytochemistry using PLZF and α6 integrin antibodies. The number and diameter of colonies of SSCs in the upper layer were evaluated at days 14 and 28 of culture. The number and diameter of colonies of SSCs were significantly higher in the melatonin group compared with the control group. The levels of expression of ID-4 and Plzf, unlike c-kit, were significantly higher in the melatonin group than in the control group. Conclusions: Results of the present study show that supplementation of the culture medium (SACS) with 100 μM melatonin significantly decreased reactive oxygen species (ROS) production in the treated group compared with the control group, and increased SSC proliferation. Keywords: Spermatogonial stem cell, Melatonin, Colonization, Three-dimensional soft agar culture system, Proliferation Background critical role in sperm preservation and management of Spermatogonial stem cells (SSCs) are germline precursor infertility, particularly among cancer survivors [3, 4]. cells with the potential to self-renew and generate differ- Recent investigations have focused on the factors entiated germ cells which have the capability of produ- associated with the proliferation of cultured SSCs, such cing progeny cells that form sperms [1]. A paucity of as leukemia inhibitory factor (LIF) [5, 6], glia cell line- these stem cells in the mature testis has limited their derived neurotrophic factor (GDNF) [5, 7], basic fibro- isolation for in vitro studies [2]. In addition, long-term blast growth factor [5, 8], and stem cell factor [9, 10]. All chemotherapy or radiation therapy in cancer patients of these factors have been found to play crucial roles in has a devastating impact on these cells and can result in the development of SSCs in vitro. However, most of these male infertility after the treatment. Therefore, methods experiments commonly investigated two-dimensional cell for efficient in vitro proliferation of SSCs could play a culture systems using culture dishes or flasks [11–13]. The main disadvantage of the two-dimensional model is the lack of metabolic and proliferative gradients similar to * Correspondence: [email protected] 1Department of Anatomy, School of Medicine, Tehran University of Medical the natural SSC niche. In a three-dimensional culture Sciences, Tehran, Iran system, Sertoli cells proliferate and create a monolayer of Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Navid et al. Stem Cell Research & Therapy (2017) 8:233 Page 2 of 10 cells which forms a feeder layer on top of which SSCs col- Isolation of neonatal testis cells onies are formed [14]. It has been shown that three- Testicular cells were obtained from 3- to 6-day-old NMRI dimensional culture systems improve cell proliferation male mice. Testes were removed from the scrotum and [15]. Some studies have also demonstrated the importance tunica albuginea was removed to obtain the testis tissue. of a three-dimensional structure on the proliferation and The testis tissues were cut into small pieces and trans- differentiation of animal and human SSCs [15–17]. The ferred to the digestion solution containing collagenase soft agar culture system (SACS) is a qualitative, three- type IV (1 mg/ml; Sigma, Germany), DNase (10 μg/ml; dimensional cell culture structure that was first used for Sigma, Germany), and hyaluronidase (0.5 mg/ml; Sigma, clonal expansion of bone marrow cells and exploration of Germany) for 20 min at 37 °C in a 5% CO2 incubator. factors that are associated with the regulation of their pro- Cells were dispersed by pipetting every 2–5 min until the liferation and differentiation [18, 19]. In 2008, Stukenborg tubules were separated. The dispersed cells were centri- et al. published a paper in which they used SACS as a fuged at 1500 g for 5 min and then washed with novel approach to study factors involved in the regulation phosphate-buffered saline (PBS). The second step of en- of the proliferation and differentiation of SSCs. This cul- zymatic digestion was carried out using the same proced- ture system provides a microenvironment similar to in ure and enzymes (15 min). The isolated cells were washed vivo conditions and mimics some aspects of the natural again with PBS [29]. three-dimensional environment to which the SSCs are ex- posed [15]. Melatonin (N-acetyl-5-methoxytryptamine) is Cell viability considered as an important hormone with antioxidant, The viability of testicular cells was assessed by immune response, cell signaling, and neuroprotective methylthiazoltetrazolium (MTT; Sigma, Germany) be- properties [20, 21]. A normal physiological metabolism of fore and after SSC culture in the SACS. Initially, the soft cells generates reactive oxygen species (ROS), especially in upper layer was removed and the cell pellet at the bot- the mitochondria, which can have adverse effects on cell tom of the 15-ml falcon tube was obtained by pipetting signaling involved in the regulation of proliferation and several times with PBS (heated to 37 °C). Following each survival [22]. Melatonin plays a critical role in reducing pipetting, centrifugation was performed at 1500 g for ROS production in cells, and inhibits a potential DNA 5 min. The isolated cells were counted using a mutation resulting from oxidative damage [23]. Similarly, hemocytometer. Finally, the cells were transferred into a Cruz et al. found that the amphiphilic nature of melatonin 96-well culture plate with each well containing a density has an important implication in the protection of mam- of 20 × 103 cells per well per 200 μl; 400 μl MEM and malian gametes and embryos from free radical-mediated 40 μl MTT were added to each well and incubated for oxidative damage and cellular death in vitro [24]. Mela- 4 h at 37 °C in a 5% CO2 incubator. Following the incu- tonin receptors (MT1, MT2) are G protein coupled recep- bation, the medium was replaced with 400 μl dimethyl tors. Several reports have indicated the expression of sulfoxide (DMSO; 1.4 M; Sigma, Germany), and the cells melatonin receptors in rat, mouse, sheep, bovine, and hu- were kept for 30 min at room temperature. The optical man Sertoli cells [25–28]. In this study, we obtained SSCs density (OD) of the plate was measured at 540 nm with from the neonate mouse using two-step enzymatic diges- the microplate reader. tion. The goal of the present study was to investigate the effects of melatonin (antioxidant) supplementation to the Intracellular ROS measurement SACS culture medium containing LIF and GDNF on the The general oxidative stress indicator CM-H2DCFDA was proliferation of neonate mouse SSCs. Our findings show used for the detection of intracellular ROS production. that SACS, along with the novel innovative medium used DCFDA (10 ml; Sigma, Life Technologies C6827) was in this study, inhibits the release of free radicals during in added to the cells and then incubated at 37 °C for 25 min. vitro culture of SSCs and provides a promising strategy The DCFDA fluorescent probe reacts with intracellular for the expansion of SSCs; the survival rate of SSCs was H2O2 to generate fluorescence emission that can be de- enhanced during a 4-week culture period using melatonin tected by flow cytometry. Measurement of intracellular as an important antioxidant in the culture medium. H2O2 production in SACS, before and after culture, was carried out by flow cytometry using DCFDA. The cells Method were washed twice with PBS and then centrifuged at Animals 2500 g for 5 min. Green fluorescence emission was mea- Three- to 6-day-old NMRI (National Medical Research sured between 500 and 530 nm using flow cytometry [30]. Institute) male mice were maintained under standard con- ditions. Animal experiments in this study were approved Flow cytometry by the ethics committee of Tehran University of Medical The efficiency and purity of the isolated SSCs were Sciences in accordance with the university’sguidelines. quantitatively assessed by flow cytometry. The two-step Navid et al. Stem Cell Research & Therapy (2017) 8:233 Page 3 of 10 enzymatic digestion protocol yielded a single-cell sus- the colonies in each well of the same group (4–6wellsin pension from 3- to 6-day-old male mice. The majority of each group). The culture medium was replaced every these cells were SSCs.
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